Nearly 5 million American are currently diagnosed with heart failure. It is now known that after an inciting cardiac insult the myocardium hypertrophies, subsequently followed by contractile dysfunction and myocardial failure. The fundamental changes in the contractile function of myocardial hypertrophy are not well understood. Valvular heart disease enables the study of human hypertrophied myocardium by obtaining myocardial biopsies at the time of valve replacement. We plan to investigate the molecular basis of altered contractile function in the most prevalent of valvular diseases - aortic stenosis, aortic insufficiency, and mitral regurgitation. Isolated myofibrils will be studied mechanically to provide a detailed characterization of altered contractile protein function within the spatial constraints of the structured myofilament lattice. Native thin filament function will be directly assessed using the in vitro motility assay in order to identify and measure the extent to which changes in thin filament function contribute to the disruption of mechanical function at the myofibrillar level. The functional contribution of phosphorylation will be determined through the mechanical assessment of myofibrils and native thin filaments before and after phosphatase treatment. Finally, mass spectrometry will be used with the specific focus of quantitating changes in phosphorylation of two regulatory proteins associated with the thin filament - troponin I and troponin T. Through this approach investigation of other post-translational changes can also be pursued as dictated by the mechanical studies. This will be the first use of cardiac native thin filaments and myofibrils to study mechano-chemical alterations of contractile proteins produced by human valvular heart disease. The goal of this study is to test the hypothesis that, during the initial stages of hypertrophy, the primary alterations in contractile function originate in thin filament proteins, and that changes in regulatory protein phosphorylation plays a major role in the progression from non-failing non-hypertrophied myocardium to end-stage failing myocardium.